RU2315312C2 - Diagnosis method for using a solution - Google Patents

Abstract

This invention provides methods for improved diagnosis of medically relevant conditions by solution based biochemical testing procedures performed in solutions of test samples. The invention provides a method to substitute the cell based morphological information contained within the cytological and/or histological data of the test sample by molecular information obtainable from the solution, wherein the original test sample is dissolved and thus enables for accurate and reproducible assessment of medically relevant diagnosis from dissolved test samples. The method according to the invention comprises the steps of determining the levels of one or more markers associated with the condition to be diagnosed, determining the level of a set of normalization markers suitable to substitute the information related to morphological aspects of the sample, that would have enabled or supported diagnosis in a cell based test system, comparing and/or combining the data concerning the levels of said markers and assessing diagnosis of a medically relevant condition.

Description

The present invention relates to methods for diagnosing medically relevant conditions by detecting levels of the corresponding markers, which are a sign of a medically relevant condition, and levels of normalization markers. These methods relate to characterization of a sample in the form of a solution without using information about cell morphology.

State of the art

Modern diagnostics of a large number of medically relevant conditions is based on the use of molecular markers as tools. Such molecular agents are usually one of the aspects of a comprehensive study and allow you to take into account a number of different parameters that characterize the studied samples.

In a medically relevant analysis, morphological examination of samples is usually carried out by cytological or histological methods. These methods are based on the morphological characterization of cell samples and are used to analyze medical samples, such as body fluids, blood, surgical sections, secrets, smears or lavage.

When screening analysis, for example, for cervical cancer, carried out in order to detect neoplastic lesions of the cervix, smears are used. In this screening procedure, lesions of various origins are detected. The causes of such lesions may be, for example, inflammation (caused by infectious agents or physical or chemical damage) or pre-tumor and tumor changes. Morphological analysis makes it difficult to distinguish between lesions of a different nature. So, for example, for the analysis of smears, cytologists and pathologists must undergo special training, and even experienced diagnostic specialists in carrying out diagnostic analyzes of cytological samples get results that are very different both from the observations of other employees and from their own observations. Generally speaking, the result of such a study is based on a subjective interpretation of the diagnostic criteria by the pathologist / cytologist conducting the study. As a result, the number of false positive and false negative results in such screening analyzes is unacceptably high.

Therefore, in many cases, such cytological or histological analyzes are confirmed using molecular markers. Such markers are often used in immunohistochemical staining reactions or in situ hybridization reactions. In the already developed combined studies on the morphology and reactions of immunohistochemical staining based on the use of marker molecules, the characterization of various medically relevant states of tissues or cells can give better results - see, for example, publication EP 1,217,377. However, morphological research is still a laborious process that requires a lot of time, and therefore is expensive even when confirmed by molecular methods, which give more reliable results. In addition, the diagnosis made on the basis of the data of cell-morphological analysis, even when confirmed by molecular parameters, depends on the subjective assessment of morphology by individual specialists. Thus, the diagnosis depends on the specialist conducting the study.

Only in very rare cases, molecular markers can be used as diagnostic tools that do not require additional confirmation by morphological examination of cells. This is especially true in cases where markers should be detected in an environment where they are found only under strictly defined conditions. Thus, diagnostic methods for medical conditions that are carried out only at the molecular level and are not supported by information about the morphology of the cell are limited only to those methods that use suitable markers that are strictly specific to the diagnosed condition. So, for example, the detection of viral infections can be carried out in sample solutions, since markers indicating the presence of viruses in tissues are not found in normal human tissues.

The reproducibility of the results of such studies can be enhanced by the application of confirmatory molecular methods. However, the problem associated with the storage and preparation of samples cannot be solved only by the additional use of molecular markers.

When using molecular methods in cytological or histological analyzes, strict precautions must be taken when storing samples to prevent the formation of artifacts and to obtain false test results. This is due in part to the lack of information on cell morphology, and partly to the instability of molecular markers detected during testing. If the preparation, transportation, or storage of samples is not properly observed, then cell information or even molecular information may be completely lost or distorted. Therefore, in this case, the diagnosis cannot be established or it may be false. For example, the interpretation of a biopsy sample or cytological preparations often presents significant difficulties or is generally impossible due to cell damage (physical or biochemical). As for tissue samples or biopsy samples, maintaining the molecular structure of the samples, which undergo rapid metabolism, is a rather difficult task, since it takes a long time to put the entire sample in the appropriate preservatives.

Diagnostic methods supported by morphological analyzes and carried out in accordance with standard procedures have two main drawbacks. First, these methods are highly dependent on the subjective assessment of the researcher. Secondly, the information obtained in morphological studies is quite sensitive to cell destruction processes, and therefore it can lead to the appearance of artifacts after obtaining samples. Both of these aspects cause poor reproducibility of the results.

To improve the diagnosis of medically relevant conditions, it is desirable to develop such methods that would not depend on the results of morphological studies of cells.

SUMMARY OF THE INVENTION

The present invention relates to a method for diagnosing a medically relevant condition of a patient. This method includes the steps of obtaining an initial sample containing cells or cell debris obtained from cells taken from a patient; obtaining a solution of the specified source sample; detecting levels of one or more relevant markers associated with said medically relevant condition in said sample solution; detecting levels of one or more normalization markers; normalization of the detected level of relevant markers according to the specified normalization parameters; and diagnosing a medically relevant condition based on normalized levels of said relevant markers in the sample solution. These normalization markers correlate with at least one of the following normalization parameters: the presence or absence of a particular type of cell among the cells present in the sample solution, the presence or absence of a specific nature of the differentiation of cells present in the sample solution; and the presence or absence of an appropriate ability to proliferate these cells present in the sample solution.

In one embodiment of the invention, said medically relevant condition is a cell proliferative disorder, cancer, or a lesion preceding it.

The present invention also relates to a test kit for diagnosing a medically relevant condition.

A brief description of the graphic material

The present patent application contains at least one illustration made in color. Copies of this patent or publication of a patent application with color illustration (s) will be provided by the Patent Office upon request and after payment of the required fee.

In FIG. 1 illustrates specific immunohistochemical staining of endocervical and ectocervical epithelial cells in cervical sections. In FIG. 1A shows a positive reaction detected in the cylindrical epithelium of endocervix (mucous membrane of the cervical canal) using an antibody directed against cytokeratin 18 (CK18). In FIG. 1B shows non-specific staining in the cylindrical epithelium of an ectocervix using an antibody directed against cytokeratin 18 (CK18). In FIG. 1C shows non-specific staining in the cylindrical epithelium of endocervix using antibodies against cytokeratin 10/13 (CK10 / 13). In FIG. 1D shows intense staining of the squamous epithelium of the ectocervix using an antibody against cytokeratin 10/13 (CK10 / 13).

In FIG. 2 illustrates Western blot analysis of solubilized samples taken from cervical smears. Numbers 1-4 refer to samples (cervical smears) obtained from individual patients.

In FIG. Figure 3 shows Western blot and ELISA assays illustrating the adequacy of the samples. Samples taken from four patients with high-grade cervical dysplasia (see "Diagnosis") were analyzed using Western blot analysis (upper part in the figure). The bottom of this figure shows the results of ELISA assays.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for improving the diagnosis of medically relevant conditions by conducting test procedures using solutions of test samples. The present invention relates to a method in which, instead of information on cell morphology obtained from cytological and / or histological analyzes of test samples, molecular information is obtained by analyzing a solution where the original test sample is dissolved and therefore suitable for accurate and reproducible analysis based on these dissolved test samples. The method according to the invention includes the steps of determining the levels of one or more markers associated with the diagnosed condition; determining the level of a series of normalization markers that can be used instead of information related to the morphological aspects of a given sample, and which should establish or confirm a diagnosis made using a cell test system; comparing and / or combining data related to the levels of these markers; and diagnosing a medically relevant condition.

The present invention indicates that the diagnosis of conditions that are usually (in cellular diagnostic systems) determined and / or confirmed by histological and / or cytological analyzes can be carried out in solutions of the original samples containing cells of various types with different properties, a method comprising the following steps : receiving the original sample; dissolving said sample in an appropriate solution; detecting the level of one or more markers associated with the diagnosed condition and, in addition, one or more normalization markers in the solution of the specified sample; normalizing data correlating with the level of markers associated with the indicated state, using data correlating with the indicated normalization markers; and diagnosing the presence or absence of this condition based on the analysis of the specified sample.

The method according to the invention can be applied, for example, for primary screening analysis in cases where a cytological, histological or pathological analysis is usually carried out. The application of the method according to the invention allows to determine whether it is possible to diagnose this condition based on the analysis of this sample. If the diagnosis based on the analysis of the solution gives a negative result regarding the presence of a particular condition, then additional analysis may be unnecessary. If a positive result was obtained, then it can be additionally confirmed by classical methods. Thus, by conducting a preliminary inexpensive and quick screening test, expensive and time-consuming microscopic analysis or other similar analyzes can be avoided.

In one of its aspects, the present invention relates to a method for improving the diagnosis of medically relevant conditions, where the diagnosis is made using solutions of the original samples of lysed tissues or cells. The diagnostic method according to the invention is based not on morphological parameters, but on biochemical analysis.

In its second aspect, the present invention relates to a method for characterizing a complex sample in solution using molecular markers that correlate with parameters of interest, which makes it possible to refuse to obtain information by conducting cytological or histological analyzes in one way or another.

In a third aspect, the present invention relates to suitable marker combinations for the diagnosis of a particular medically relevant condition using complex samples. Markers for normalization are chosen so that from the initial sample it was possible to determine parameters that would help to make or confirm an appropriate diagnosis and which could replace the data lost when the sample was dissolved.

In its fourth aspect, the present invention relates to a test kit for establishing a diagnosis or for conducting research in accordance with the present invention.

The present invention makes it possible to quickly and easily diagnose this condition by analyzing an initial sample, such as physiological fluids, smears, lavage (e.g. bronchoalveolar lavage, lactic duct lavage, etc.), aspirates (aspirates taken with a needle; aspirates taken with a thin needle ) or complex cellular or tissue samples. Generally speaking, the problem associated with the use of the original samples is that other types of cells, as well as specific microorganisms and extracellular substances, are present in this sample. Thus, the starting material contains a mixture of cells and compositions, which can lead to the formation of artifacts. The presence of cells of various types with different proliferative properties, as well as various microorganisms and substances in the original sample, leads to the appearance of many factors that can affect a particular level of marker molecules. Thus, the detection of the level of only one single molecular marker provides diagnostically useful information if, in addition, the (morphological) parameters related to the original sample are determined. All morphological data obtained from the original sample are lost due to lysis of cells in solution. However, there are suitable molecular markers that correspond to specific morphological or other parameters obtained by histological and cytological methods.

So, for example, information about one component in the original sample can be obtained in the classical way by examination under a microscope. Morphological research provides information on the differentiation and localization of cells, as well as on the environment of these cells. For example, specific cells in cytological preparations of cervical smears can be identified as epithelial cells and, in addition, they can be classified as endocervical or ectocervical epithelial cells. By examining under the microscope, even the presence of non-cervical cells, such as endometrial cells, can be easily detected.

In accordance with the present invention, the starting materials can be directly dissolved in an appropriate solvent, without additional preparation or characterization, regardless of the homogeneity or heterogeneity of the starting material. Data whose loss is due to the lysis of the material contained in the sample solution is determined by the levels of a series of marker molecules, and therefore can be restored using the indicated molecular data to normalize according to the corresponding morphological parameters. This can be achieved using an appropriate series of molecular markers for each of the characteristic parameters necessary to establish an accurate diagnosis. By detecting the corresponding array of markers, the corresponding parameters characterizing the given initial sample can be evaluated, which avoids undesirable loss of information due to lysis of the sample.

The test procedure according to the invention includes the detection of levels of markers, which are signs of the considered cellular conditions, and markers to normalize data by parameters characterizing the specific environment of the test sample. Markers suitable for use in the present invention may have a different origin. The nature of the expression of a marker suitable for the detection of the conditions under consideration may depend on the proliferative status of cells, on the status of differentiation of cells, on the type of cells or on the type of organism. Examples of suitable markers are described below.

As used herein, the term “diagnosis” essentially means any analysis for the presence or absence of a medically relevant condition. For example, the term “diagnosis” includes procedures such as screening for a predisposition to a medically relevant condition, screening for a harbinger of a medically relevant condition, screening for a medically relevant condition and a clinical or pathological diagnosis of a medically relevant condition, and the like. As used herein, the term “diagnosis of a medically relevant condition” may include evaluating any condition detected at a cytological, histological, biochemical or molecular biological level, and may refer to human health and / or body. Such studies may include, for example, tests performed by medical diagnostic methods, and scientific research in the field of biology. In one embodiment of the invention, such a method is used to diagnose medically relevant conditions, such as diseases. Such diseases may be, for example, disorders characterized by proliferation of wild-type cells or tissues.

In one embodiment, the term “diagnosis” refers to the diagnosis of cancer and precancerous conditions, to the monitoring of the course of a cancer, to the prognostic evaluation of cancer, and to the detection of disseminated tumor cells, for example, in the process of establishing a diagnosis of minimal residual disease. The method according to the invention can, for example, be used for the clinical or pathological diagnosis of cancer and precancerous conditions or in routine screening tests performed to detect a specific cancer, such as, for example, smear tests, for example, screening tests for cervical lesions analysis of bronchial lavage for lung cancer, or analysis for damage to the gastrointestinal tract, for example, for damage to the rectum and colon.

The method according to the invention can be applied to all types of medically relevant conditions.

As used herein, the term “medically relevant conditions” may, for example, include the composition of tissues, body fluids, secretions, washes or smears. The term “condition” may, for example, mean the cellular composition of physiological fluids, such as blood composition, fluid composition, or sperm composition. In this context, the term “composition” will mean, for example, the presence or absence of specific types of cells (for example, pathogens such as viruses and the like; preneoplastic, neoplastic and / or dysplastic cells, etc.), the presence or absence the specific nature of the differentiation of cells of a particular type, the total number of cells of a particular type (for example, red blood cells, white blood cells, sperm cells, etc.), the total number of all cells of any type or fraction of cells with other properties that are present or absent in this sample.

In addition, the term “medically relevant conditions” may also include disorders related to cells or tissues. The term "diagnosed conditions" may include parameters related to cells in cytological or histological tissue samples. The term “conditions” may include the specific nature of the differentiation of cells in a tissue sample, such as samples of surgical sections, biopsies, smears, lavages, and the like. Such conditions may be, for example, hereditary disorders, inflammatory diseases, mechanical disorders, injuries, vascular diseases, degenerative disorders, growth disorders, benign tumors and malignant tumors. In another aspect of the invention, such disorders may be those characterized by the presence or absence of proliferative properties. Conditions characterized by the presence or absence of proliferative properties may be, for example, cell proliferative disorders.

Cell proliferative disorders according to the invention include diseases characterized by the ability of cells or tissues to abnormally grow compared to normal growth of control cells or tissues. The growth of such cells or tissues may, for example, be abnormally accelerated, reduced, or it may undergo abnormal regulation. As used herein, the term "abnormal regulation" can mean any form of the presence or absence of a cellular or tissue non-wild type response to natural processes that influence growth regulation. The term "cell or tissue growth abnormalities" may mean, for example, neoplasia or hyperplasia.

In one embodiment, cell proliferative disorders are tumors. The term “tumors” may include head and neck tumors, airway tumors, anogenital tract tumors, gastrointestinal tumors, urinary system tumors, reproductive system tumors, endocrine system tumors, central and peripheral nervous system tumors, skin tumors and their derivatives, tumors of soft tissues and bones, tumors of the lymphopoietic and hematopoietic system, etc. The term “tumors” can also mean neoplasms, such as benign and malignant tumors, carcinomas, sarcomas, leukemias, lymphomas or dysplasias. In a particular embodiment, such tumors are, for example, head and neck cancer, respiratory tract cancer, cancer of the anogenital tract, cancer of the gastrointestinal tract, cancer of the skin and its derivatives, cancer of the central and peripheral nervous system, cancer of the urinary system, cancer of the reproductive system , cancer of the endocrine system, cancer of soft tissues and bones, cancer of the lymphopoietic and hematopoietic system, etc.

Tumors of the anogenital tract may include perineal cancer, perineal scrotal skin cancer, cervical cancer, vulvar cancer, vaginal cancer, penis cancer, anus cancer, and the like. Cervical cancer may include squamous lesions, glandular lesions, or other epithelial tumors. Squamous lesions include, for example, intraepithelial cervical tumors (mild, moderate and significant dysplasia), in situ carcinoma, squamous cell carcinoma (keratinizing, non-keratinizing, papillary, oncoplastic, papillary and lymphoepithelial carcinomas). Glandular lesions may include atypical hyperplasia, in situ adenocarcinomas, adenocarcinomas (e.g., mucus-forming, endometrioid, transparent cell adenocarcinoma, malignant denoma, papillary, serous or mesonephric adenocarcinoma). Other epithelial tumors may include adenosquamous carcinoma, vitreous cell carcinoma, adenocystic carcinoma, adenobasal cell carcinoma, carcinoid tumor, small cell carcinoma, and undifferentiated carcinoma. For more information, see "Kurman R., Norris H. et al., Tumors of the Cervix, Vagina and Vulva, Atlas of Tumor Pathology, 1992, AFIP", the contents of which are incorporated into this description by reference.

Tumors of the gastrointestinal tract may include colon cancer, cancer of the ascending colon, descending colon, transverse colon, sigmoid colon, colon cancer, small intestine cancer, colon cancer, duodenal cancer, gastric cancer, cancer esophagus, liver cancer, cancer of the biliary tract, cancer of the biliary system, cancer of the pancreas, etc. A comprehensive description of gastrointestinal lesions is provided in Hamilton Sr., Aaltonen LA (Eds.): World Health Organization Classification of Tumors, Pathology and Genetics of Tumors of the Digestive System, LARC Press: Lyon 2000, which is being introduced description by reference.

The term “airway tumor” can mean any malignant disease of the airway, such as, for example, lung cancer, alveoli, bronchioles, bronchial tree and bronchi, nasopharyngeal cancer, oral cancer, laryngeal cancer, nasal cavity and paranasal sinus cancer. Lung cancer includes small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma of the lung, small cell carcinoma of the lung, adenocarcinoma of the lung, large cell carcinoma of the lung, squamous adenocarcinoma of the lung, carcinoid tumor of the lung, tumor of the bronchial gland or (malignant) mesoma. A general description of airway tumors can be found in Colby T.V. et al .: Tumors of the Lower Respiratory Tract, Atlas of Tumor Pathology, Third Series, Fascicle 13, AFIP: Washington 1995 ", which is incorporated herein by reference.

Tumors of the urinary system may include bladder cancer, kidney cancer, renal pelvis cancer, ureter cancer, urethral cancer, and the like. Tumors of the reproductive system may include cancer and precancer of the ovary, uterus, testes, prostate gland, epididymis, etc.

In all cases, the methods of the invention can also be used to treat precancerous lesions, tumors or cancer.

In one embodiment, the method of the invention comprises the detection of disseminated tumor cells or metastases.

In one embodiment, the carcinoma is, for example, cervical cancer, colon cancer, stomach cancer, breast cancer, bladder cancer, lung cancer, oral cancer, and the like.

The present invention relates to a number of reliable, quick and easy ways to preserve the molecular properties of samples and thereby prevent the loss of morphological information that these samples contain. Such samples can, for example, be obtained in reproducible and convenient for storage and transportation form by dissolving the cellular components of the original sample in a suitable solvent immediately after receipt of the sample or even during its preparation. Body fluids can be directly transferred from the body of the individual to a solution containing suitable detergents and preservatives. In addition, tissue samples can be directly transferred to denaturing lysis conditions (actually supported by physical forces) and thus they can be stored. The molecular components of the original sample can be stored using the appropriate ingredients in a solvent, and therefore they cannot be degraded. The degradation of enzymatic activities can, for example, be minimized using enzyme inhibitors. Thus, in the solution of test samples, the molecular properties of the test sample can easily be preserved during dissolution, and no additional storage conditions are required.

The term "source samples" may include clinical samples, such as secrets, smears, lavage, physiological fluids, blood, urine, sperm, feces, bile and fluid samples, bone marrow samples, biopsy specimens and samples of cells and tissues. The term "biopsy" as used in the context of the present invention may include, for example, sections of tumors, tissue samples taken by endoscopic methods or by trepanobiopsy or puncture (aspiration) organ biopsy. In addition, the sample according to the invention can be any sample that potentially contains detectable marker molecules. In one embodiment, said sample includes cervical smears, bronchial lavage, feces, and the like. The term "source sample", as used in the context of the present invention, may include fixed or stored samples of cells or tissues. So, for example, cells stored in suitable solutions (alcohols, etc.) or fixed tissue samples can be used as starting samples in the methods of the invention.

The original sample used in the method according to the invention is any sample containing cells or cell debris. Such cells may be, for example, prokaryotic or eukaryotic cells.

If the present invention relates to the detection of infectious diseases, then the detected cells may be cells of microorganisms, such as chlamydia, E. coli , Candida , etc.

In accordance with the present invention, all or part of the molecular components of the starting samples are solubilized in a suitable lysis buffer containing, for example, solvents. Such solvents may be, for example, aqueous solutions of chaotropic agents, such as urea, GuaSCN, formamide, aqueous solutions of detergents, such as anionic detergents (e.g. SDS, N-lauryl sarcosine, sodium deoxycholate, alkylarylsulfonates, sulfates of long chain (fatty) alcohols, and olefin sulfonates, sulfates and sulfonates of alpha olefins, sulfonated monoglycerides, sulfonated esters, sulfosuccinates, alkanesulfonates, phosphoric esters, alkyl isethionates, sucrose esters), cationic e detergents (e.g. cetyltrimethylammonium chloride), nonionic detergents (e.g. Tween-20, Nonidet P-40, Triton X-100, NP-40, Igepal CA-630, N-octyl glucoside) or amphoteric detergents (e.g. CHAPS, 3 -dedecyldimethylammonium propane-1-sulfonate, lauryl dimethylamine oxide) and / or aqueous solutions of alkali metal hydroxides, such as, for example, NaOH or KOH. The solvent is chosen so that cells, cell debris, nucleic acids, polypeptides, lipids, and other biomolecules that may be present in the original sample can dissolve in it. The solution for dissolving the original samples according to the invention may, in addition, contain one or more agents that prevent the degradation of the components in these initial samples. Such components may be, for example, enzyme inhibitors, such as proteinase inhibitors, RNase inhibitors, DNase inhibitors, and the like. In one embodiment, the sample is lysed in the form in which it was directly taken from the individuals being examined. In another embodiment, the sample may also be purified and then lysed. Such cleaning procedures may, for example, include removal (by washing) of impurities such as mucus or the like, isolation or concentration of cellular components, preservation and transportation of cells. So, for example, the cellular components of the original samples can be included in a single sample solution.

Obtaining a sample for use in the method described herein may also include several additional steps for preparing the sample, such as separating insoluble components, isolating polypeptides or nucleic acids, preparing peptides or nucleic acids fixed on the solid phase, or obtaining spheres, membranes or slides with which defined molecules can covalently or non-covalently bind.

In accordance with the present invention, the detection of marker molecules is carried out directly in this solution. Such detection can be carried out in solution or using reagents fixed on the solid phase. In some embodiments, marker molecules are detected in a solution of physiological samples dissolved therein. Therefore, such detection can be carried out in solution or using reagents fixed on the solid phase. The solid phase used in the context of the present invention can be various types of solids, such as flat surfaces and particles (including micro- and nanoparticles or even smaller particles). In some embodiments, such particles may be spheres, colloids, or the like. The fixation of reagents on the solid phase in the test kit or in the device for in vitro diagnostics can be carried out by direct or indirect fixation. Direct fixation can, for example, be accomplished by covalent or non-covalent binding or association with surfaces. Indirect fixation can be accomplished by binding reagents (e.g. antibodies, probes, etc.) to agents that are themselves directly fixed on solid phases. Such agents may be antibodies or other binding agents, such as streptavidin, biotin, or the like. The detection of one or more molecular markers can be carried out in a single reaction mixture or in two or in several separate reaction mixtures. Detection reactions of several marker molecules can, for example, be carried out simultaneously in multi-well reaction vessels or, in this case, it can be carried out on one, two or more separate test plates. Markers that are signs of cell proliferative disorders can be detected using reagents that specifically recognize these molecules. At the same time, normalization markers can be detected using reagents that specifically recognize these markers. The detection reaction of markers of each class may include one or more additional reactions with detecting agents that either recognize the source marker molecules or preferably recognize the precursor molecules (eg, “first” antibodies) used to recognize the source markers. The detection reaction, in addition, may include a "reporter" reaction, indicating the level of markers, which are a sign of cell proliferative disorders or markers of normalization.

The terms "marker" or "marker molecule", as used in the context of the present invention in all grammatical forms, mean nucleic acid as well as polypeptide molecules. For example, the terms “marker” or “marker molecule” mean, for example, RNA (mRNA, hRNA, etc.), DNA (cDNA, genomic DNA, etc.), proteins, polypeptides, proteoglycans, glycoproteins and corresponding fragments of these molecules. The term “relevant marker” means marker molecules that are indications of a medically relevant condition. The term "normalization marker" means marker molecules used for normalization purposes.

As used herein, the term “marker molecule level” means a semi-quantitative as well as a quantitative quantity related to the amount of the corresponding marker present in the sample. A quantitative value may, for example, be defined as concentration. The semi-quantitative value can be expressed as a scale of levels, for example, undetectable levels, low levels, intermediate levels, high levels, or any other suitable method. The marker level can also be represented as a dependent parameter, such as the intensity of the signal generated in the analysis format in response to the presence of the marker molecule.

The probe for detecting marker molecules used in the present invention may be any molecule that specifically binds to said marker molecules. Such a probe may be, for example, an antigen binding agent, such as antibodies (monoclonal or polyclonal antibodies), antibody fragments or artificial molecules containing antigen binding epitopes, DNA or RNA binding molecules, such as proteins or nucleic acids. Nucleic acids that bind to other nucleic acids can be, for example, peptide-linked nucleic acids (PNA), oligonucleotides (RNA, DNA, PNA, artificial nucleic acids, etc.) used for detection, or primers.

It is said that a molecule recognizes other molecules if it specifically interacts with these molecules. Such a specific interaction may be, for example, the specific binding of this molecule to another molecule.

A “reporter” reaction may be, for example, a reaction that produces a color of the compound. In one embodiment of the invention, said “reporter” substances associated with particular markers produce various color reactions. In another embodiment, the “reporter” specific for the normalization marker may be a molecule that quenches the signal produced by the reporter molecule specific for the marker associated with this medically relevant condition, depending on the level of the normalization marker present in the sample. In yet another embodiment of the invention, “reporter” reactions can produce fluorescent dyes emitting at different wavelengths. In yet another embodiment of the invention, such a reporter reaction may be the emission of light at different wavelengths characteristic of these reporter substances specific to any detectable marker. In another embodiment of the invention, such a reporter reaction may be radioactive radiation and other pathways providing visualization or quantification of the radiation. In one embodiment, various marker molecules can be recognized by agents carrying radionuclides emitting at different energy levels, whereby signals detecting marker molecules can be recognized.

The formats used for the detection reaction according to the invention can be blotting methods such as Western blot analysis, Southern blot analysis and Northern blot analysis. Blotting methods are known to those skilled in the art and can be implemented, for example, in the format of electro-blotting, semi-dry blotting, vacuum blotting or dot blotting. In addition, immunological methods, such as, for example, immunoprecipitation or immunological analyzes, such as ELISA, ELISA, RIA, side-flow analysis, flow cytometry analysis, immunochromatographic plate analysis, etc., can be used to detect molecules. The immunoassays used in the present invention may include both competitive and non-competitive immunoassays.

In some embodiments of the invention, testing carried out by immunochemical methods or methods using nucleic acids can be carried out using a test device for clinical laboratories. Such a test device can be any device suitable for testing carried out by immunochemical methods or methods of any format using nucleic acids, for example, accurate test instruments for testing, as well as desktop or laboratory devices. These devices can also be equipped with open or closed systems such as platforms. These systems can be developed on the basis of any suitable technique, such as, for example, the use of microtiter plates, multi-well plates, through-stream or side-stream systems, microchips or matrix systems, or systems using spheres or membranes. The detection methods used may be any methods known to those skilled in the art that are commonly used to carry out immunochemical detection reactions or detection reactions at the nucleic acid level. Such detection systems can be, for example, luminescent systems (electroluminescent, bioluminescent, photoluminescent, radioluminescent, chemiluminescent, electrochemiluminescent systems), systems based on fluorescence, systems based on conductivity, systems based on irradiation with UV-visible light x-ray radiation, gamma radiation, etc.), or systems based on the use of any other known method.

In one embodiment, the marker molecular level detection method is any method suitable for detecting even a very small number of specific molecules in biological samples. In addition, any method for detecting marker molecules can be applied regardless of its sensitivity. The detection reaction according to the invention may include, for example, nucleic acid level detection reactions and / or polypeptide level detection reactions. In one embodiment, the detection of marker molecules may include the detection of specific splicing variants. In another embodiment of the invention, said detection method may include detecting modifications of marker molecules such as phosphorylation, glycosylation, or the like. polypeptides, or methylation of nucleic acid molecules in samples.

In one embodiment of the invention, the detection of the level of marker molecules is carried out by determining the level of nucleic acids encoding the marker molecules or fragments thereof present in the sample. Methods for detecting nucleic acid molecules are known in the art. The nucleic acid detection procedure can be carried out, for example, by the reaction of binding of the detected molecule with complementary nucleic acid probes, with proteins having binding specificity with nucleic acids, or with any other molecules that specifically recognize these nucleic acids and bind to them. Such a method can be carried out in vitro, as well as directly in situ, for example, during the detection process carried out by means of a staining reaction. Another method for detecting marker molecules in a sample at a nucleic acid level carried out in the method according to the invention is a nucleic acid amplification reaction that can be carried out quantitatively, for example, polymerase chain reaction. In one embodiment of the invention, a quantitative assessment of the level of marker RNA in samples for the diagnosis of cell proliferative disorders can be carried out, for example, using RT-PCR in real time.

In another embodiment, the detection of the level of marker molecules is carried out by determining the level of expression of the protein. Detection of marker molecules at the protein level can be carried out, for example, in a reaction mixture containing a binding agent specific for the detection of marker molecules. Such binding agents may be, for example, antibodies and antigen binding fragments, bifunctional hybrid antibodies, peptidomimetics containing a minimal amount of antigen binding epitopes. These binding agents can be used in many different detection methods, such as, for example, Western blotting, ELISA, RIA, ELISA, flow cytometry, side-stream analysis, latex agglutination method, immunochromatographic plate assay and immunoprecipitation. Typically, detection using binding agents can be carried out in vitro, as well as directly in situ, for example, in the course of an immunocytochemical staining reaction. Any method suitable for determining the amount of specific polypeptides in solutions of biological samples may be used in the present invention.

Methods for detecting modified nucleic acid molecules and / or polypeptides are known in the art.

Detection methods for nucleic acid methylation are known to those skilled in the art, and such methods can be, for example, methods using preliminary chemical treatment, for example, sodium bisulfite, permanganate or hydrazine, followed by detection of this modification using specific restriction endonucleases or specific probes, for example, during the amplification reaction . Methylation detection can also be carried out using restriction endonucleases responsible for methylation. Methods for detecting nucleic acid methylation are described, for example, in patent applications EP02010272.9, US 5856094, WO 0031294, US6331393 and the like. Cited documents are incorporated herein by reference.

Detection of modifications in polypeptides can, for example, be carried out using binding agents that specifically recognize modified or unmodified forms of the polypeptides. Alternatively, enzymes such as phosphatases or glycolases can be used to remove modifications in the molecules. Thus, the presence or absence of modifications can be detected by determining the mass or charge of the molecules by electrophoresis, chromatography, mass spectrometry, etc. before or after incubation with the appropriate enzyme.

In yet another embodiment, the detection of a series of markers can be carried out at a polypeptide level, and at the same time, a series of marker molecules and / or all or some of these marker molecules can be detected at a nucleic acid level.

Markers associated with medically relevant conditions can be, for example, molecules that influence and / or reflect the nature of the proliferation and / or differentiation of cells and / or tissues. Such molecules may include, for example, cell cycle regulation proteins; proteins associated with DNA replication; transmembrane proteins, receptor proteins; signal transducing proteins; calcium binding proteins; proteins containing DNA-binding domains; metalloproteinases; kinases; kinase inhibitors; chaperones; embryogenesis proteins; heat shock proteins or enzymes that provide post-translational modification of other proteins and thereby regulate their activity; or nucleic acids encoding these proteins. Marker molecules used in the present invention may also be mRNAs encoding these proteins. In one embodiment of the invention, the marker associated with a cell proliferative disorder may, for example, be uniquely expressed in affected cells, but may not be expressed in these cells or overexpressed in these cells.

The marker molecules used in the present invention may include one or more markers selected from p13.5, p14, p15, p16 (also referred to as p16 ^INK4a ), p19, p21, p27, p53, pRb, p14ARF, cyclin A, cyclin B , cyclin E, MDM-2, MCM2, MCM5, MCM6, CDC2, CDC6, Id1, osteopontin, GRP, renal dipeptidase, her2 / neu, TGFβII receptor, HPV-associated markers, for example, derived from the HPV, L1, L2 genes , E1, E2, E4, E5, E6 or E7, etc. The selection of markers that can be used in one of the embodiments according to the invention for the detection of medically relevant conditions is illustrated below in table 1.

In one embodiment, the marker for the medically relevant condition may be a marker associated with tumors (tumor markers). Marker molecules associated with tumors can be, for example, proteins that undergo non-wild type expression, in contrast to normal control tissue. As used herein, the term "non-wild type expression" may mean increased or decreased expression levels, lack of expression, or expression of the corresponding molecules in non-wild type forms. The expression of a non-wild-type protein may include expression of mutated forms of proteins having mutations caused by insertion, deletion, substitution or frame shift, or expression of proteins or nucleic acids with other known types of mutations. In all cases of expression of non-wild-type proteins or levels of non-wild-type proteins, proteins, polypeptides or fragments thereof or nucleic acids encoding these proteins, polypeptides or fragments thereof can be used as molecular markers associated with tumors, and such thus may fall within the concept of "tumor marker" as used in the context of the invention. Proteins that exhibit non-wild type expression associated with tumors are described, for example, in documents WO 9904265A2, WO 0149716A2, WO 0055633A2 and WO 0142792A2, which are incorporated herein by reference.

In one embodiment of the invention, the marker, which is a sign of a medically relevant condition, may be a cell cycle regulation protein, such as, for example, cyclin, cyclin-dependent kinase, or a cyclin-dependent kinase inhibitor. In another embodiment, the marker that is a sign of a medically relevant condition may be a marker associated with a transient or persistent viral infection. Such a viral infection may be an infection caused by human papillomavirus (HPV), such as high or low risk HPV. High-risk HPV may include HPV subtypes such as, for example, HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 58. Markers for HPV infection can be, for example, HPV gene expression products such as L1, L2, E2, E4, E5, E6 or E7. In a third embodiment of the invention, a marker associated with a viral infection can be used in combination with any other marker for a medically relevant condition, for example, in combination with a cell cycle regulation protein. Combinations of marker molecules that may be of particular interest in terms of their association with HPV are described, for example, in WO 0208764, which is incorporated herein by reference.

In one embodiment, cell cycle regulatory proteins used in combination with HPV markers can be selected, for example, from the group consisting of pRb, p53, p14ARF and cyclin-dependent kinase inhibitors. In one specific embodiment, for example, p16 ^INK4a can be used in combination with markers for HPV infection (for example, L1, L2, E2, E4, E5, E6 or E7).

It should be noted that in this case, markers, which in some embodiments of the invention are used as markers of medically relevant conditions, in some other embodiments of the invention can serve as normalization markers and vice versa. However, in each hotel embodiment, the marker can serve as either a marker of a medically relevant condition or a marker of normalization. So, for example, in some embodiments, Ki67, used as a marker for cell proliferation, can be used as a normalization marker (for example, in combination with p16, p14ARF, claudin-1, or other markers of a medically relevant condition); in other embodiments, Ki67 may serve as a marker for a medically relevant condition (e.g., a marker for cervical dysplasia or other dysplastic diseases) in combination with appropriate normalization markers (e.g., cytokeratins, catenins, or the like). Various other markers can also serve either as a marker for medically relevant conditions or as a normalization marker, depending on the particular application.

In accordance with the present invention, normalization markers may be, for example, “housekeeping” genes, such as actin, gapth, histone, phospholipase, β2-microglobulin, proteins associated with active cell proliferation, such as Ki67, PCNA or statin, or proteins characteristic of specific cell types, such as CK20 in epithelial cells, or any cell-specific antigens of the cell surface. In addition, carbohydrate structures present on glycoproteins, proteoglycans, lectin receptors, such as the concanavalin A receptor, mucins, and enzymes that are involved in the biosynthesis of these molecules, such as GalNac transferases and oligosaccharyl transferases, can also serve as normalization markers. The type of marker protein is selected depending on the information provided by the indicated marker. In principle, the markers used to identify specific medically relevant conditions can, in certain cases, be used as normalization markers. The selection of markers suitable for implementing the methods of the invention is illustrated in Table 1.

As for markers of medically relevant conditions, as well as markers of normalization, molecules in a modified form (such as polypeptides and nucleic acids) can be used as such markers in the method according to the invention. So, for example, in the method according to the invention, phosphorylated, glycosylated or otherwise modified polypeptides or methylated nucleic acids can be used as markers.

The term “normalization” as used in the present invention means any method suitable for determining the relationship between detectable levels of markers and parameters necessary to establish a diagnosis. In one aspect of the invention, such a normalization may be the restoration of the corresponding cytological and histological information contained in the original sample using suitable molecular markers detected in sample solutions. Such normalization may include, for example, determining the total number of cells present in the sample; the presence or absence of cells of a particular type in the sample; the presence or absence of microorganisms or their cells in the sample; the number of cells of a particular type or cells of the microorganism present in the sample, the proliferative properties of the cells present in the sample; or the nature of the differentiation of cells present in the sample.

In some embodiments of the invention, normalization may also include confirmation of the adequacy of the test, where in this case, inadequate test results may either not be taken into account or may be classified as invalid. Therefore, the term "normalization", as used in the context of the invention, can mean qualitative or semi-quantitative methods of normalization. In some embodiments, semi-quantitative normalization may include determining a threshold value for the normalization marker. In one embodiment, semi-quantitative normalization can be used for the following interpretations of the results: the level determined for a given relevant marker can be considered as a true test result only if the level of such a normalization marker exceeds a certain threshold value; if the specified threshold value is not reached, then the test result for the relevant marker is considered incorrect, and the diagnosis cannot be established on the basis of this test. In other embodiments, a threshold value that cannot be exceeded can be set. In some embodiments of the invention, qualitative normalization can be carried out by the presence or absence of a normalization marker. In these cases, for example, the value determined for the relevant marker is compared with the value obtained in the presence or absence of a normalization marker. As previously determined, the value is considered true only if the normalization parameter was set (the presence or absence of a detectable level of the normalization marker).

Table 1 Marker for Cell type Antigen Antibody Provider Literature cell type epithelial cells Human Epithelial Cell Surface Glycoprotein NEA125
IgG1 (W, IHC, ICC, IF) Research Diagnostics Inc. Kommoss et al., Hum Pathol. 2000 Sep; 31 (9): 1055-61 40 kD protein associated with proliferation of human epithelial cells (from LoVo) AUA-1
IgG1
(Elisa) Research Diagnostics Inc. Gottschalk et al., Pathol Res Pract. 1992 Feb; 188 (1-2): 182-90 Human Epithelial Cell Antigen
(34 + 39 cd) Ber-EP4, IgG1 (IHC, Elisa) Dako Latza U et al., J Clin Pathol. 1990 Mar; 43 (3): 213-9 Antigen Associated with the Proliferation of Human Epithelial Cells (40 kD) AUA-1 (Elisa, W, IHC) Research Diagnostics Inc. Epenetos, A et al., Lancet. 1982 Nov 6: 2 (8306):
1004-6 Cytokeratin 18 (45 kD) RGE 53, IgG1 (W, IHC, IF) Research Diagnostics Inc. Smedts F et al., Am J Pathol. 1990 Mar; 136 (3): 657-68 Endocervical cylindrical cells Cytokeratin 18 (45 kD) RCK 106 (W, IHF, IHC) Research Diagnostics Inc. Smedts F et al., Am J Pathol. 1990 Mar; 136 (3): 657-68 Cytokeratin 8 (52.5 kD) CAM 5.2 (W, IHC) BD PharMingen Smedts F et al., Am J Pathol. 1990 Mar; 136 (3): 657-68 Endocervical cylindrical cells Mucin antigens (Tn, STn, MUC1, MUC2) Df3 Centocor Tashiro et al., Hum Pathol. 1994 Apr: 25 (4): 364-72 Endocervical cylindrical cells Concanavalin A receptor Herckenrode et al., Br J Cancer. 1988 Mar; 57 (3): 293-4; Koch et al., Br J Cancer. 1986 Jan; 53 (1): 13-22 Endocervix GalNac transferase
Oligosaccharyltransferase Chilton et al., Endocrinology. 1988 Sep; 123 (3): 1237-44 Endocervix / Ectocervix Lectins (ConA, WGA, PNA, UEAI, DBA, SBA, SNA Di Loretto et al., Basic Appl Histochem. 1987; 31 (2): 143-52;
Versura et al., Basic Appl Histochem. 1988; 32 (2): 219-27 ectocervix squamous cells Placophilin (80.5 kD) PP1-5C2, IgG1 (W, Elisa, IHC, IF) Research Diagnostics Inc. Heid. HW, Differentiation. 1994 Dec; 58 (2): 113-31 Endometrial cells Vimentin VIM 3B4, IgG1, (W, ELISA, IF, IHC) Research Diagnostics Inc. Smedts F et al., Am J Pathol. 1990 Mar; 136 (3): 657-68 Red blood cells Hemoglobin RDI-CBL63, IgM, (RIA, EIA) Research Diagnostics Inc. Smith et al., J. Histochem. Cytochem. 1998 CD16 (NK, Macro, Gran) DJ130-c, IgG1 (IHC) DIANOVA Grundhoever D and Patterson BK, Cytometry 2001; 46: 340-344 Neutrophils
Granulocytes CD56 (NK) Clone Ki-M6 Hermann et al., J. Clin. Immunol. 1990 Inflammation NK cells
Macrophages CD68 (Macro) (against CD68) Cavayal et al., Eur. J. Immunol: 1998 (6) 1991-2002 (CD56) B cells CD19 (CD20) Clone AE1
Facs DIANOVA Harrada et al., Blood 1993; 81: 2658-63 (CD19) Mason et al., Am J. Pathol 1990; 136: 1215-22 (CD20) T cells CD3 (pan-T cells) (CD4);
(Cd8) Clone CRIS-7 (against CD3); IF, IHC, WB DIANOVA Jones et al., J Immunol 1993; 150: 5429-35 dysplastic and neoplastic cervical cells p16 ^INK4a E6H4, D7D7 MTM Klaes R., et al. Int J Cancer. 2001 Apr 15; 92 (2): 276-84 cancer cells of various types P53 (mutations) Mendoza-Rodriguez CA, et al., Rev Invest Clin 2001 May-Jun; 53 (3): 266-73 Tumor cells Adenocarcinoma cells CEA Mistretta et al., Experientia. 1974 Oct 15; 30 (10): 1209-10;
Rogers et al., Eur J Cancer Clin Oncol. 1984 Oct; 20 (10): 1279-86 Bladder cancer cells NMP22, BTA Van der Poel HG et al., Curr Opin Urol, 11, 503-509, 2001 Lung cancer cells PreproGRP Hamid et al., Cancer, 63, 266-271, 1989, Pagani et al., Int. J. Cancer 47, 371-375, 1991 Proliferation all proliferating cells PCNA Ki67 Pc10, IgG2a Zymed Wassem NH, Lane DP, J Cell Sci 1990: 96: 121 (PCNA) Cattoretti et al., J Pathol 1992: 168: 357-63 (Ki67) HPV 16 E6 BF7, IgG1 (IHC and in diagnostic kits for cervical smears) Research Diagnostics Inc. Iftner et al., J Virol. 1988 Oct; 62 (10): 3655-61 L1 CamVir-1, IgG2a (IP, W, IF, IHC) Research Diagnostics Inc. Browne L et al., J Gen Virol. 1988 Jun; 69 (Pt6): 1263-73 Infectious agent HPV 18 L1 RDI-HPV18-5A3, IgG1 (W, IHC) Research Diagnostics Inc. Iftner et al., J Virol. 1988 Oct; 62 (10): 3655-61 HPV 6,11,18 RDI-HPVX-4C4 Research Diagnostics Inc. Iftner et al., J Virol. 1988 Oct; 62 (10): 3655-61 Gouillou et al., Am. J. Surg. Pathol., 1991

In accordance with the present invention, normalization may include determining the presence of a certain number of considered cells (human) in a sample. This is a very important aspect of the present invention. For example, obtaining false (in particular, false negative) test results can be avoided if the test procedure confirms that the test sample contains the materials (for example, cells, tissues, microorganisms, etc.) necessary for a specific test. In various tests, this will mean ensuring that the sample contains cells. With a broad examination of embodiments of the invention, confirmation of the adequacy of this sample involves not only confirmation of the presence of cells, but also detection of the presence of cells of various nature or cells of a certain type.

Thus, normalization may include the detection of cells of a specific origin, such as, for example, cells of a particular organ or a specific histological localization, for example, the detection of cells of different regions of the epithelium, connective tissue cells, cells derived from the basement membrane of the tissue, or cells of heterologous origin, such metastatic cells. In specific cases, this may be necessary, because among them there may be cells that, under certain conditions, express a marker that can be used to detect a medically relevant condition, such as, for example, neoplasia or dysplasia, in some normal conditions. Thus, the term “normalization” as used in the present invention can mean the detection of the presence or absence and / or determination of the level of cells of any type, which may contribute to the overall level of specific markers selected for the diagnosis of a medically relevant condition.

In one embodiment, the method can be used to detect cervical lesions. Damage to the cervix can mean cervical dysplasia of any type, such as cervical cancer, as defined above, and its preceding stages. Markers and their combinations used for detection purposes are described, for example, in documents WO 0208764 and EP1217377, which are incorporated herein by reference. In this embodiment of the invention, said test can be carried out using any suitable sample taken from the cervix (cervical sample). Such samples can be, for example, biopsy specimens or microbiopsy specimens taken from the cervix, or smears taken from the cervical region. Cervical smears used herein are samples that can, for example, be obtained using a suitable device, such as a brush, swab, spatula, or the like, that come into contact with the cervix during the sample collection procedure. The sampling device may be any suitable device that can be used in standard testing procedures performed by a physician or automatically by the sampling device itself.

Promising molecular markers that can be used to increase the efficiency of analysis of cervical samples are, for example, p16 ^INK4a , p14ARF, cyclin E, cyclin A, cyclin B, MN, her2 / neu, mdm-2, bcl-2, EGF receptor , mcm-2, mcm-5, claudin-1, markers indicative of human papillomavirus infection, pRb, p53, etc., which can be used to detect dysplastic and neoplastic cells. In accordance with the present invention, the normalization used to analyze cervical smears may include detection of the presence of all human cells, detection of cervical epithelial cells, detection of the presence of endocervical as well as ectocervical cells, and detection of cells of endometrial origin. The endocervical epithelium is a glandular cylindrical epithelium. Cells originating from endocervix can be identified by markers that are selectively expressed by cylindrical epithelial cells or glandular epithelial cells. Ectocervical epithelium is squamous epithelium. Ectocervical cell identification can be achieved by detecting markers associated with squamous epithelial cells. In some embodiments, the detection of epithelial cells (containing squamous as well as cylindrical epithelium) may be sufficient. In other embodiments, the differentiation of endocervical cells may be a very important parameter. This important stage confirms the presence of ecto- and endocervical cells in this sample, that is, confirms that this sample was taken from the transformed zone of the cervix, where the greatest dysplasia and neoplasia are observed. If such cells are absent, then this sample is not suitable for the testing procedure, that is, it can give false negative results. Since p16 ^INK4a can be expressed in normal endometrial cells, it may be necessary to normalize the expression levels of p16 ^INK4a by the number of endometrial cells.

Therefore, for a reliable diagnosis, normalization may include detection of the presence or absence of the aforementioned cellular components in the sample, and, in addition, detection of the general level of cells of a particular type or their fraction, which make a certain contribution to the total number of cells present in the sample.

Thus, in one embodiment of the invention, the detected level of p16 ^INK4a protein can be normalized by the cytological conditions available in this particular sample, so that it can be determined whether the detected level of p16 ^INK4a protein is indicative of overexpression of p16 ^INK4a in cervical cells, or this sample contains an excessive number of endometrial cells, which can simulate overexpression of p16 ^INK4a . In this regard, normalization may include determining the number of endometrial cells in the cervical sample based on molecular marker levels. By comparing the level, for example, of p16 ^INK4a , used as a marker of a medically relevant condition determined in a cervical sample, with the number of endometrial cells assessed using molecular markers, it can be determined whether the entire amount of p16 belongs only to the endometrial cells present in this sample solution . Thus, in the diagnosis of cervical dysplasia, false-positive results obtained due to the fact that elevated p16 ^INK4a levels due to the presence of high levels of endometrial cells are erroneously attributed to overexpression of p16 ^INK4a in cervical cells can be ruled out. The term "quantity" as used in the context of the present invention may mean a quantitative or semi-quantitative assessment. This term may, for example, mean an estimate of the total number of cells or an estimate of the cell fraction with respect to the total number of cells. In some embodiments of the invention, the term "quantification" may mean the evaluation of the fraction of the total level of markers, which is provided by cells of a particular type.

In order to obtain a normalization marker for analysis of cervical samples, several normalization markers can be used and these markers can be selected, for example, from cytokeratins, E-cadherins, involucrin, urokinase-like plasminogen activator, SCCA (squamous cell carcinoma antigen), catenins (e.g. alpha catenin, beta-catenin, gamma-catenin (placoglobin)), EP-Cam.

Some candidates for normalization markers have been examined in terms of their ability to characterize cervical specimens. The results are presented in table 2 and table 3.

Normalization markers can, for example, be used as indicators of the presence of cells with a specific nature of differentiation, for example, terminal differentiation or differentiation specific for epithelial cells. In some embodiments, normalization markers can serve as marker molecules characteristic of squamous epithelial cells, for example, as indicators of the presence of ectocervical cells in a cervical sample. Suitable markers include, for example, CK13, E-cadherin, gamma-catenin, or involucrin. In another embodiment, such markers can serve as indicators of the presence of cylindrical epithelial cells, indicating the presence of endocervical cells in a given sample. Suitable markers are: Ep-Cam, CK18, CK8.

In some embodiments, normalization may include detection of substantially epithelial cells, and any marker suitable for detecting epithelial cells may be used for these purposes. Such markers may be, for example, markers described in tables 2 and 3.

In yet another embodiment of the invention, the method described herein can be used to detect respiratory disorders. One of the markers used to diagnose small cell lung cancer is neuron-specific enolase (NSE). Tumor samples are obtained by bronchoscopy with the collection of cells using brushes or bronchoalveolar lavages. Since NSE is also expressed in some normal lung cells, the level of NSE expression detected in the dissolved sample must be determined relative to the level of the normalization marker (e.g. actin) to detect the number of cells present in the sample.

In a third embodiment, the present invention relates to the detection of lesions of the gastrointestinal tract, for example, the detection of lesions of the colon and rectum by analysis of feces. In this case, the origin of indicator nucleic acids and / or polypeptides detected in fecal samples can be crucial for the diagnosis. In accordance with the present invention, the origin (cell types / microorganisms) of the marker molecules used can be determined. Thus, a false-positive result based, for example, on the detection of marker molecules derived from food digested by the individual, and not from lesions of the gastrointestinal mucosa, can be ruled out. In addition, using the technique described in this application, artifacts arising from the presence of trace amounts of markers coming from the bloodstream or from swallowed sputum, etc. can be excluded.

In another aspect, the present invention relates to a test kit for implementing the method according to the invention. This kit may be, for example, a diagnostic kit, an analytical kit, or a research kit.

The term “kit” as used in the present invention may include kits as well as diagnostic devices. Such kits can be, for example, kits intended for analysis: ELISA (for example, “sandwich” analyzes, competitive or non-competitive analyzes, etc.), ELISA (competitive or non-competitive) or RIA; test systems using spheres, end-to-end or side-flow systems, analytical test plates, or any other known laboratory, bench-top or precision test instrumentation. In some embodiments, a kit according to the invention may include in vitro diagnostic devices for performing diagnostic tests. Such in vitro diagnostic devices may be any type of ELISA device known to those skilled in the art. Such devices may include devices for analysis in the sandwich-ELISA format, in the competitive ELISA format, and in any other ELISA assay formats. In another embodiment, the in vitro diagnostic devices may be devices for side-stream analysis or end-to-end flow analysis (flow cytometry), carried out, for example, using at least one reagent that binds to an indicator marker of a medically relevant condition, and one reagent that binds to a normalization marker, both of which are fixed on the solid phase. In such devices, various mechanisms for visualizing test results can be used. In some embodiments of the invention, “second” detection reagents directed against marker molecules associated with the detected molecules can be used in these tests. Such detectable molecules can be colloidal gold (colored), latex particles, and other molecules.

In yet another embodiment, the in vitro diagnostic test device may be a flow analysis device based on the use of capillary or porous membranes (such as membranes, spheres, or other three-dimensional structures of porous substances). Depending on the embodiment of the present invention, the size of the pores or capillaries must be adjusted in order to ensure optimal conditions for flow analysis.

A kit according to the invention may include:

a) reagents for the detection of marker molecules,

b) reagents and buffers that are commonly used to carry out the detection reaction, such as buffers, detectable markers, carriers, etc .;

c) one or more markers and / or samples representative of the diagnosed medically relevant conditions and used to carry out positive and / or negative control reactions, and

d) one or more samples with normalization markers for positive and / or negative control reactions,

Such a test kit may, but not necessarily, include a lysis buffer to solubilize the original sample. In general, any suitable solvent known to those skilled in the art can be a lysis buffer. The lysis buffers used in this kit can be, for example, aqueous solutions of chaotropic agents such as urea, GuaSCN, formamide, aqueous solutions of detergents such as anionic detergents (e.g. SDS, N-laurylsarcosine, sodium deoxycholate, alkylarylsulfonates, sulfates long chain (fatty) alcohols, olefin sulfates and sulfonates, alpha olefin sulfates and sulfonates, sulfonated monoglycerides, sulfonated esters, sulfosuccinates, alkanesulfonates, phosphoric esters, alkyl isethionates, complex sucrose esters), cationic detergents (e.g. cetyltrimethylammonium chloride), nonionic detergents (e.g. Tween-20, Nonidet P-40, Triton X-100, NP-40, Igepal CA-630, N-octylglucoside) or amphoteric detergents ( for example, CHAPS, 3-dodecyldimethylammonium propane-1-sulfonate, lauryl dimethylamine oxide) and / or aqueous solutions of alkali metal hydroxides, such as, for example, NaOH or KOH.

Examples of lysis buffers are presented in table 4.

The lysis buffer, in addition, may contain one or more agents that prevent the degradation of components in the original samples. Such components may, for example, contain enzyme inhibitors, such as proteinase inhibitors, RNase inhibitors, DNase inhibitors, and the like. Such inhibitors may be, for example, proteinase inhibitors selected from the compositions shown in Table 5. In some embodiments, lysis buffers due to the presence of a degradation inhibitor can detect p16 in a given sample. In some embodiments, the cyclin-dependent kinase p16 inhibitor is degraded in solubilized samples, and therefore cannot be detected. This is especially true when the samples are directly transferred to the lysing medium and stored there for a certain period of time.

Table 5 Inhibitor Inhibitable Proteinase Class Concentration Solubility in water Water stability Aprotinin Serine 0.6-2 mcg / ml Very good Good Benzamidine Serine 0.5-4 mm good good Bestatin aminopeptidase 1-10 μM good good Calpeptin Cysteine 0.3-1 μM good good Cystatin Cysteine 1 μM good good E-64 Cysteine 1-10 μM good good EDTA Metalloproteinase 0.5-5 μM good good Elastatin Serine 0.5-2 mcg / ml good good Est Cysteine 20-50 mcg / ml good bad Veal Fetal Serum all classes 10% good good Leipeptin Serine / Cysteine 10-100 μM good good a2-macro-globulin all classes 1 μM good good Nco-700 Cysteine 0.5-100 μM bad bad Pefabloc =
AEBSF Serine 0.2-10 μM good very bad Pepstatin A Aspartic 1 μM bad Bad PMSF Serine 0.2-10 μM bad very bad o-phenanthroline Metalloproteinase 1-10 mm bad Bad

The lysis buffer used for stabilization may also contain bulk protein (e.g., albumin, such as bovine serum albumin or calf serum albumin or other bulk proteins) to prevent protein degradation of the sample. Bulk proteins may, for example, be present in combination with proteinase inhibitors, or they may be added instead of proteinase inhibitors. In one embodiment of the invention, the solvent may be selected so that it is compatible with the conditions of the test (ELISA, ELISA or analysis on the plates) and that solubilized samples can be directly used in this test. The term "test", as used in the context of the present invention, means any procedure performed to detect the presence or absence of marker molecules and / or determine their levels.

The reagent used to detect marker molecules may include any agent capable of binding to the marker molecule. Such reagents can be proteins, (poly) peptides, nucleic acids, peptide-linked nucleic acids (PNAs), glycoproteins, proteoglycans, polysaccharides or lipids.

Samples including markers associated with medically relevant conditions and / or normalization markers used for positive and / or negative control may contain, for example, nucleic acids in a suitable form, such as a solution or salt, peptides in a suitable form, section samples tissues, microorganisms, and positive or negative cell lines.

In one embodiment of the invention, the detection of marker molecules is carried out at the level of polypeptides. In this embodiment, the binding agent may be, for example, an antibody specific for said marker molecules, or fragments thereof. In addition, the binding agents may be antigen binding fragments, such as Fab fragments, single chain antibodies, bifunctional hybrid antibodies, peptidomimetics containing minimal antigen binding epitopes and the like. In addition, the binding agent may be a lectin that binds to a specific carbohydrate structure on the marker molecule.

In another embodiment of the test kit, the detection of marker molecules is carried out at the level of nucleic acids. In this embodiment, such a detection reagent may be, for example, a nucleic acid probe or a reverse primer complementary to said marker nucleic acid.

The following examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention.

Examples

Example 1: Specific immunohistochemical detection of endocervical and ectocervical epithelial cells in cervical sections

To evaluate markers indicating the adequacy of cervical smears, cervical sections (fixed in a 4% formaldehyde solution and embedded in paraffin) were stained with anti-cytokeratin 18 antibody (marker for endocervical cylindrical epithelium) and anti-cytokeratin 10/13 antibody (marker for ectocervical squamous). In FIG. 1 shows specific staining of endocervical epithelium with anti-cytokeratin 18 antibody and specific staining of ectocervical epithelium with anti-cytokeratin antibody 10/13. This experiment was carried out as follows:

Formalin-fixed and paraffin-embedded sections were subjected to dewaxing in a xylene bath for 5 minutes (the step was repeated one more time), the excess liquid was removed and the slides were placed in 95-96% ethanol for 3 (± 1) minutes, then in 70% ethanol for 3 (± 1) minutes (the stage was repeated one more time), and finally in distilled water for at least 30 seconds. To restore the epitope, slides were placed in a Koplin vessel and boiled for 40 minutes at 95-99 ° C in 10 mM citrate buffer, pH 6.0. Then the slides were left in this buffer for 20 minutes (± 1 min) to cool to room temperature. After that, the slides were coated with a peroxidase blocking reagent (3% H ₂ O ₂ ; 15 mM NaN ₃ ) and incubated for 5 (± 1) minutes at room temperature. After a 5-minute wash in wash buffer, the slides were incubated with the “first” antibodies (SK 10/13: DE-K13, 1:50, DAKO; SK 18: K18.7, 1 μg / ml, Dianova) for 30 minutes . The slides were then rinsed with wash buffer and washed in wash buffer for 5 minutes at room temperature. After a 30-minute incubation with EnVision (with the ready-to-use anti-mouse antibody-horseradish peroxidase complex; DAKO), the slides were washed 3 times for 5 minutes and incubated in a DAB substrate for 10 minutes, then subjected to contrast staining with hematoxylin and transferred to a support environment (Faramount).

Using an anti-cytokeratin 18 antibody (SK 18) in an immunohistochemical staining procedure, a positive reaction was observed in the cylindrical endocervix epithelium (Fig. 1A), while the squamous ectocervix epithelium did not show specific staining (Fig. 1B). When immunohistochemical staining with an antibody directed against cytokeratin 10/13 (CK 10/13), staining of the endocervix cylindrical epithelium was not detected (Fig. 1C), but intense staining of the squamous epithelium of ectocervix was observed (Fig. 1D). Therefore, CK18 can be used as a specific marker for the detection of cylindrical epithelial cells of endocervix, and CK10 / 13 can be used as a specific marker for the detection of squamous epithelial cells of ectocervix.

Example 2: Western blot analysis of solubilized samples obtained from cervical smears

In order to determine whether it is possible to diagnose cervical lesions using Western blot analysis of solubilized samples, clinical samples taken from a patient with an established diagnosis were subjected to immunochemical analysis based on detection of marker molecules after lysis of the starting material.

Samples of clinical material (cervical smears) were analyzed using standard Western analysis as follows.

Briefly, in the first stage, the clinical material was solubilized by boiling (5 min, 95 ° C) in a Laemmli buffer for protein samples (100 mM Tris, pH 6.8, 2% SDS, 200 mM DTT, 0.05% VRB), and then treated with ultrasound. In the second stage, protein samples were separated by SDS-PAGE (12% acrylamide) by electrophoresis and then transferred onto a nitrocellulose membrane by blotting in a tank (Towbin et al., 1979, Proc. Natl. Acad. Sci .: 76: 4350- 4354). At another stage, membranes were blocked to prevent non-specific binding to the antibody (in 10% skimmed milk powder in PBS), and then incubated with a specific monoclonal mouse antibody (SK 8: 35βH11, 1: 100, DAKO; p16 ^INK4a : D7D7, 1: 140 , MTM Laboratories). The binding of a specific antibody was visualized using “second” reagents conjugated to horseradish peroxidase (binding to a marker-specific antibody) and catalyzing photon-emitting substrates.

Cytokeratin 8 (SK 8) was used as a marker specific for endocervical cells, the presence of which indicated the adequacy of sample collection in the experiments according to the invention. An inhibitor of cyclin-dependent kinase p16 ^{INK4a was} used as a disease-specific marker.

The experimental results of the present invention are shown in FIG. 2. Numbers 1-4 refer to samples (cervical smears) taken from individual patients. Immuno-blot detection was performed using specific antibodies against cytokeratin 8 (CK8) and specific antibodies against p16 ^INK4a (p16). Samples taken from patients 1, 2, and 3 did not show any signal on p16 ^INK4a . This means that dysplastic cervical cells are absent in these samples. A sample taken from the patient 4, gave an intense signal for p16 ^INK4a. This means that dysplastic cervical cells are present in this sample. The upper bands showed specific signals for cytokeratin 8. In samples 1, 3, and 4, cytokeratin 8 could be detected, while no signal was detected in sample 2. This indicates that endocervical cylindrical cells are present in samples 1, 3 and 4, but are absent in sample 2. Since the presence of endocervical cylindrical epithelial cells is one of the indicators of the adequacy of cervical smears, then sample 2 was considered as inadequate, and if the result of detection on p16 ^INK4a was negative then, in this case the diagnosis cannot be established. Samples 1, 3 and 4 were considered as adequate. Therefore, based on the negative signal of samples 1 and 3 on p16 ^INK4a, we can conclude that these patients did not have cervical dysplasia. Sample 4 gave a positive signal to p16 ^INK4a , which indicated that the patient had dysplastic lesions of the cervix.

A parallel cytological analysis of smears indicated normal cell composition in women 1 and 3. In woman 2, the diagnosis was not made due to a lack of cellular material. Woman 4 was diagnosed with high-grade dysplasia. It should be noted that the upper band (SC 8) corresponds to a normalization marker specific for endocervical cells, namely cytokeratin 8, which indicates the adequacy of sample collection. The lower band refers to the marker p16 ^INK4a, associated with a particular disease. This blot for patient 4 gave a positive signal to p16 ^INK4a , indicating that she had a high-grade cervical dysplasia. Samples taken from patients 1 and 3 showed only a CK8-specific band, which indicated a “correct” collection of samples, but did not indicate the presence of a marker associated with this disease (p16 ^INK4a ) and corresponded to normal, healthy cervical epithelium. The sample for patient 2 did not detect the CK8 signal, which corresponded to the low number of cells in this sample, and therefore, if the result of detection on p16 ^INK4a was negative, then in this case the diagnosis could not be established.

Example 3: Western Blot Analysis and ELISA Assay Conducted to Demonstrate Sample Adequacy

In order to determine whether the differences between the results of the diagnosis established on the basis of the analysis of the solution and the results of the diagnosis established on the basis of the analysis of the samples can be explained by the inadequacy of the sample, Western blot analysis of cervical smears taken from four different patients with a diagnosis (diagnosis of high-grade cervical intraepithelial neoplasia, in accordance with the cytological diagnosis of Pap IVa and Pap IVb). An anti-p16 ^INK4a antibody was used to detect the presence of dysplastic cells, and anti-CK18 and CK 10/13 antibodies were used to demonstrate the adequacy of the sample.

Western blot analysis was performed as follows: samples taken from patients were collected with a cervical brush and directly lysed in Laemmli sample buffer (2% SDS, 60 mM Tris, pH 6.8, 0.01%, 100 mM DTT) in for 5 minutes at 95 ° C (1 x 10 ⁷ cells / ml), and then treated with ultrasound (5 pulses for 5 seconds, maximum intensity). Lysates were centrifuged for 12 minutes at 16,600 x g in a microcentrifuge, and the supernatant was transferred to a new tube. Pre-prepared gels in a linear gradient of 4-20% acrylamide (Criterion System, Bio-Rad) were loaded with 10 μl (10 ⁵ cells) of whole cell extracts, and the proteins were separated by a constant electric current of 25 mA for 45 minutes. Proteins were transferred from the gel to a Hybond ECL nitrocellulose membrane (Amersham) by standard blotting in a tank using a Bio Rad Criterion Blotter blotter (15 minutes at a constant voltage of 100 Volts, and then 45 minutes at a constant voltage of 50 Volts). Nitrocellulose membranes were stained for 5 minutes in a Ponceau S solution to confirm protein transfer. The Ponceau S solution was removed by two 10-minute washes in PBS. For immunodetection, blots were blocked overnight in blocking buffer (10% milk powder in PBS with 0.1% Tween-20). The "first" antibodies were incubated at dilutions recommended by the manufacturer in a blocking buffer for 1 hour at room temperature with stirring (CK18: MAB 3236), 1: 1000, CHEMICON; SC 10/13: DE-K13, 1: 500, DAKO, p16 ^INK4a : D7D7, 1: 140, MTM Laboratories). After six 10-minute washes with PBS / 0.1% Tween, 20 blots were incubated with rabbit anti-mouse antibody conjugated with HRP (DAKO diluted 1: 5000 in blocking buffer) for 1 hour at room temperature. After six 10-minute washes with PBS / 0.1% Tween, 20 membranes were incubated for 5 minutes in a substrate solution (Super Signal West Femto Maximum Substrate, Pierce), wrapped in plastic and exposed to X-ray film for 1-5 minutes. Finally, X-ray films were developed, fixed, dried and read using a visualization system (Bio-Rad). The same samples were used for ELISA analysis on p16 ^INK4a , SK 10/13, SK18. The detected signals and results were similar to the results of Western blot analysis, based on which the final conclusions were made.

ELISA analysis was performed as follows. Flat-bottom 96-well plates (MaxiSorb; Nunc) were sensitized with an immobilized antibody (p16 ^INK4a : MTM-E6H4, 2 μg / ml in PBS, MTM Laboratories; CK10: MS481P1ABX, 2 μg / ml, dianova; CK18: K18.7.2 μg / ml, Dianova; 50 μl / well) overnight at 4 ° C. The plates were washed 6 times with PBS / 0.1% Tween-20 and blocked with Superblock buffer (Pierce). The solubilized protein extract isolated from cervical smears was dissolved in incubation buffer (PBS, 3% Superblock, 0.1% Tween-20) and added to each well in triplicate. After incubation for 1 hour at room temperature the plates were washed 6 times with PBS / 0,1% Tween-20 and incubated with biotinylated antibody for detection (p16 ^INK4a: MTM-D7D7 (0,2 g / ml, MTM Laboratories, CK10: MS481 -BO, 200 μg / ml, Dianova; CK18: MS142-BO, 200 μg / ml, Dianova; in incubation buffer) for 1 hour at room temperature After six washes with PBS / 0.1% Tween-20 TMB 50 μl of alkaline phosphatase bound to streptavidin (1: 1000 dilution, Dianova) was added over 30 minutes, then the plates were washed 6 times with PBS / 0.1% Tween-20 and 100 μl of sub was added to each well p-nitrophenyl phosphate (PnPP; dissolved in diethanolamine buffer). OD 405 nm (comparable wavelength 620 nm) was measured on an ELISA reader (Tecan) after 30 minutes, 1 hour and 2 hours. This example showed that the analysis in the format " -ELISA sandwich showed a sensitivity suitable for use in the methods of the invention. The sandwich-ELISA analysis used in the method of the invention described in this example can be applied to a variety of marker molecules, such as normalization / adequacy markers and markers that are signs of medically eligible states.

Samples taken from four patients with a diagnosis of high-grade cervical dysplasia (see "Diagnosis") were analyzed using Western blot analysis (top panel in the figure). For the left blot, immunoblot detection was performed using antibodies specific for β-actin and p16 ^INK4a , for the middle blot, using antibodies specific for cytokeratin 10/13, and for the right blot, using antibodies specific for cytokeratin 18. B β-actin, CK18 and CK 10/13, which demonstrated the adequacy of the sample, were used as markers. β-actin indicated the presence of any cells, CK10 / 13 indicated the presence of ectocervical squamous cells, and CK18 indicated the presence of endocervical cylindrical cells.

As shown in FIG. 3, for samples taken from patients 1 and 2, immunoblots showed positive signals for all used adequacy markers (CK10 / 13, CK18, β-actin) and for the marker (p16 ^INK4a ), which is an indicator of the presence of dysplastic cells. Samples 3 and 4 in the Western blot analysis were negative for p16 ^INK4a bands. However, in these Western blot analyzes, β-actin and two cytokeratin markers gave an extremely weak signal (patient 3, β-actin) or no signal at all (patient 4, all markers; patient 3, SK markers). Therefore, based on the absence of a signal on p16 ^{INK4a, the} diagnosis cannot be established.

The bottom panel of this figure shows the results of ELISA analyzes. Positive signals for adequacy markers (CK10 / 13, CK18) were detected for a sample taken from patients 1 and 2, while for samples taken from patients 3 and 4, signals to CK10 / 13 and CK 18 were not observed. Thus, the results of ELISA analysis coincided with the results of Western blot analysis, based on which similar conclusions were made.

Example 4: Western blot analysis of various samples taken from the lungs

In order to determine whether it was possible to diagnose lung lesions using Western blot analysis of solubilized samples, clinical samples taken from a patient with an established diagnosis were solubilized and subjected to immunochemical analysis based on marker molecules and normalization molecules.

Clinical samples (cells collected by brush or bronchoalveolar lavage) were analyzed using standard Western analysis as follows. Cells collected by bronchoalveolar lavage were pelleted by centrifugation (5 minutes, 1000 rpm), and the pellet was dissolved in Laemmli buffer for protein samples (100 mM Tris, pH 6.8, 2% SDS, 200 mM DTT, 0.05 % VRB). Cells collected using a brush were directly dissolved in Laemmli buffer for protein samples (100 mM Tris, pH 6.8, 2% SDS, 200 mM DTT, 0.05% VRB). This material was boiled (5 min, 95 ° C) and then treated with ultrasound. In the second stage, aliquots of protein samples in duplicates were separated by SDS-PAGE (12% acrylamide) and then transferred onto a nitrocellulose membrane by blotting in a tank (Towbin et al., 1979, Proc.Natl. Acad. Sci .: 76 : 4350-4354). At another stage, membranes were blocked to prevent non-specific binding to the antibody (in 10% skimmed milk powder in PBS), and then one membrane was incubated with specific anti-NSE monoclonal mouse antibodies (DAKO Germany, clone BSS / NC / VI-H14, mouse monoclonal antibody (diluted 1: 1000), and the other membrane was incubated with a normalization marker, actin (ICN, USA, clone C4, mouse monoclonal antibody, diluted 1: 400). The binding of a specific antibody was visualized using “second” reagents conjugated to horseradish peroxidase (binding to a marker-specific antibody) and catalyzing photon-emitting substrates.

In bronchoalveolar lavages of patients with a diagnosis of small cell lung cancer, high levels of NSE were detected compared to levels of expression of actin, while in patients without tumors, almost no NSE was found, however, the levels of actin in these patients were comparable to the level of actin in patients with cancer. (Data not shown).

These results showed that the normalization of the test procedure carried out in solution in accordance with the method described here, allows you to establish a diagnosis of diseases without using data from morphological studies.

Example 5: Detection of cervical intraepithelial neoplasia in an ELISA test

34 cervical smear placed in a lysis buffer were subjected to ELISA-detecting overexpression of an inhibitor of cyclin-dependent kinase inhibitor p16 ^INK4a in solutions prepared from the cells contained in these smears. An ELISA test was performed as described below.

(A) Lysis of cells

Cervical smear brushes were placed in 15 ml vessels containing 2 ml mtm lysis buffer. Cervical cells present on the brush were lysed for at least 20 hours. Then, lysates of cervical smear samples were transferred to 2 ml tubes and centrifuged at 4 ° C (15 minutes at 28,000 x g (16,600 rpm, JEC Multi RF High Speed Centrifuge)). The supernatant was transferred to a clean tube. In this case, the supernatant can be stored at -20 ° C.

(B) Implementation of ELISA

Sensitization of ELISA tablets

Stock solutions of clone p16 ^INK4a- specific antibody mtm E6H4, clone Ep-Cam-specific antibody Ber-Ep4 and clone antibody 15 against gamma-catenin were diluted in PBS to obtain a ready-made sensitization solution.

50 μl of each prepared solution used for sensitization with an immobilized antibody was added to ELISA plates.

For sensitization, the plates were incubated overnight at 4 ° C.

Sensitization solutions were removed from ELISA plates, and the plates were rinsed in an automatic ELISA plate washer using 7 x 250 μl wash buffer (0.1% Tween 20 (v / v) in PBS).

After removing residual wash buffer, 300 μl blocking buffer (2% BSA in PBS) was added to each well. The plates were incubated for 1 hour on a shaker at ambient temperature.

Sample Incubation

After blocking buffer was removed, 100 μl of a sample of lysed cells was added to each well. HeLa cell lysates were used as a positive control for antibodies specifically recognizing p16 ^INK4a and gamma-catenin; HT29 cell lysates were used as a positive control for antibodies specifically recognizing Ep-Cam.

For the calibration of the test, various concentrations of recombinant p16 protein, recombinant gamma-catenin and Ep-Cam (0 pg / ml, 50 pg / ml, 100 pg / ml, 200 pg / ml, 400 pg / ml, 800 pg) were used in this test. / ml).

Samples were incubated for 1 hour at room temperature.

Then, washing was carried out in an automatic device for washing ELISA plates using 7 x 250 μl wash buffer. Residual buffer was removed.

Incubation with antibody for detection

Working solutions of biotinylated "second" antibodies (clone of the mtm D7D7 antibody specific for p16 ^INK4a , the ^A5B4 antibody clone specific for Ep-Cam, and clone MAB 2083 specific for gamma-catenin) were obtained by diluting the mother liquors.

100 μl of working solutions of biotinylated "second" antibodies were added to the wells incubated with the corresponding antigen and immobilized antibody. After incubation for 1 hour at room temperature, the antibody solutions were removed and the ELISA plates were washed in an automatic ELISA plate washer using 7 x 250 μl wash buffer.

Detection

The streptavidin-HRP polymers (1 mg / ml) were pre-diluted 1:10 (4 μl + 36 μl incubation buffer). A final incubation solution was prepared by diluting 1: 300 in an incubation buffer (0.1% BSA in PBS) to a final concentration of 0.33 μg / ml.

100 μl of the resulting solution was added to each well and incubated for 1 hour at room temperature.

The buffer was then removed and the plates were washed 5 times manually with 200 μl wash buffer per well.

Substrate incubation

TMB substrate was balanced to 25 ° C for 1 hour in the dark.

100 μl of substrate solution was added to each well.

ELISA plates were incubated at 25 ° C in the dark for exactly 15 minutes. Then the reaction was stopped by adding 80 μl of 2.5 M H ₂ SO ₄ .

5 minutes after termination of the reaction, OD 450 nm was determined. After evaluating the results, the OD value for each sample was again determined.

Evaluation of the results

To establish the adequacy of the samples, the OD values of all samples for gamma-catenin must exceed a certain threshold value, which will confirm the “correct” collection of samples containing a minimum number of cells. In addition, the threshold value for the OD-value of EP-Cam, indicating the presence of endocervical cells, should be higher to confirm "correct" sample collection.

To detect dysplastic cells, the OD values for p16 ^INK4a must exceed a certain threshold value, which will confirm the presence of a minimum number of p16-positive dysplastic cells.

The results of this experiment are shown in table 6.

Table 6 Sample No. p16 ^INK4a Gamma-catenin Conclusion 3 + + The sample is adequate; p16 ^INK4a indicates the presence of dysplastic cells thirty - + The sample is adequate; no detectable levels of p16 ^INK4a indicates the absence of dysplastic cells one - - The sample is inadequate; re-collection of samples required

Comparison of OD values for p16 ^INK4a and gamma-catenin obtained for 34 samples with the corresponding threshold levels revealed that 33 samples were adequate and could be further investigated. Of these 33 samples, 30 samples were negative for p16 ^INK4a , and 3 samples were positive.

The results of ELISA analysis were compared with the diagnostic results obtained using the Papanicolaou test (PAP test, cervical cytology) for the same patients. A cervical cytological test was performed in accordance with Munich Classification II (1990). Rar II covers benign cells, cells of the cervicitis and metaplasia region, Rar IV covers significant dysplasia and carcinoma in situ. It was found that the samples that gave OD for p16 ^INK4a over 0.9 in ELISA assay corresponded to samples which, in accordance with a standard cytological PAP test were classified as dysplastic.

Assuming OD = 0.9 as the threshold for evaluating samples in an ELISA, the following results were obtained (see Table 7).

Table 7 Diagnosis / ELISA Results p16 ^INK4a positive ELISA results p16 ^{INK4a -} negative ELISA results Rar II 0 thirty Rar IV 3 0 Insufficient number of cells 0 one

The ELISA test was positive for all 3 samples (100%) taken from women with significant dysplasia, and negative for all 30 samples (100%) taken from women without dysplasia. And only one sample contained very few cells, and therefore it was excluded from the study, since its collection was inadequate.

Normalization of p16 ^INK4a protein levels in solubilized patient samples by normalization markers indicating the presence of epithelial cells allows the diagnosis of dysplasia to be established based on analogues of these samples. In this case, such a normalization eliminates false negative results due to inadequate collection of samples (for example, if the total amount of material received from the patient is insufficient for analysis, or if this material was not collected from the desired anatomical site). This normalization was carried out in a test format using the OD threshold for the gamma-catenin normalization marker defined in the ELISA as described above, and the sample taken was classified as adequate. If the OD value was below a certain threshold value (corresponding to 200,000 squamous ectocervical cells), then it was concluded that this sample does not contain an adequate amount of material taken from the patient. The use of a second normalization marker indicated the presence of endocervical cells and provided additional information regarding the adequacy of the sample. This normalization was carried out in a test format using the OD threshold for the EP-Cam normalization marker defined in the ELISA as described above, and the sample taken was classified as adequate. If the OD value was below a certain threshold value (corresponding to 2000 cylindrical endocervical cells), then it was concluded that this sample does not contain an adequate number of endocervical cells. (It should be noted that the threshold value used in this example was adjusted according to the specific reaction conditions. This value for these cells, as well as for OD, may vary depending on the reaction conditions. Thus, the values used here are given only to illustrate the conditions of the test and should not be construed as limiting the scope of the invention, and each specialist can independently determine the appropriate threshold value for a particular test format). The presence of endocervical cells provides information about the contact of the smear or brush with the cylindrical epithelium of the endocervix, and therefore indicates the contact of the smear or brush with the transformation zone, in which cervical dysplasia usually occurs. In particular, the detection of a certain number of ectocervical cells (gamma-catenin) together with a certain number of endocervical cells (Ep-Cam) gives highly reliable information that this material was taken in the desired anatomical site (cervical transformation zone).

Using the thresholds determined in these experiments, cytological samples taken from 300 patients were tested in an ELISA format analysis. In these experiments, samples identified as dysplastic in a cytological assay can also be identified as dysplastic in an ELISA format assay.

Example 6: Detection of cervical intraepithelial neoplasia in the format of an ELISA test

34 cervical smears already used in the experiment described in Example 5 and placed in the lysis buffer were subjected to ELISA for overexpression of HPV E7 protein and one adequacy marker in solutions obtained from cells contained in these smears. An ELISA test was performed as described below.

(A) Lysis of cells

Cervical smear brushes were placed in 15 ml vessels containing 2 ml mtm lysis buffer. Cervical cells present on the brush were lysed for at least 20 hours. Then, lysates of cervical smear samples were transferred to 2 ml tubes and centrifuged at 4 ° C (15 minutes at 28,000 x g (16,600 rpm, JEC Multi RF High Speed Centrifuge)). The supernatant was transferred to a clean tube. In this case, the supernatant can be stored at -20 ° C.

(B) Implementation of ELISA

Sensitization of ELISA tablets.

Stock solutions of the E7-specific antibody clone NM2 and the anti-gamma-catenin antibody clone 15 were diluted in PBS to give a ready-made sensitization solution.

50 μl of each prepared solution used for sensitization with an immobilized antibody was added to ELISA plates.

For sensitization, the plates were incubated overnight at 4 ° C.

Sensitization solutions were removed from ELISA plates, and the plates were rinsed in an automatic ELISA plate washer using 7 x 250 μl wash buffer (0.1% Tween 20 (v / v) in PBS).

After removing residual wash buffer, 300 μl blocking buffer (2% BSA in PBS) was added to each well. The plates were incubated for 1 hour on a shaker at ambient temperature.

Sample Incubation

After blocking buffer was removed, 100 μl of a sample of lysed cells was added to each well. HeLa cell lysates were used as a positive control for antibodies specifically recognizing gamma-catenin. To calibrate the test, various concentrations of recombinant HPV 16 E7 protein, recombinant gamma-catenin (0 pg / ml, 50 pg / ml, 100 pg / ml, 200 pg / ml, 400 pg / ml, 800 pg / ml) were used in this test. .

Samples were incubated for 1 hour at room temperature.

Then, washing was carried out in an automatic device for washing ELISA plates using 7 x 250 μl wash buffer. Residual buffer was removed.

Incubation with antibody for detection

Working solutions of biotinylated "second" antibodies (clone NM13 specific for HPV16 E7 protein and clone MAB 2083 specific for gamma-catenin) were obtained by diluting the mother liquors.

100 μl of working solutions of biotinylated "second" antibodies were added to the wells incubated with the corresponding antigen and immobilized antibody. After incubation for 1 hour at room temperature, the antibody solutions were removed and the ELISA plates were washed in an automatic ELISA plate washer using 7 x 250 μl wash buffer.

Detection

The streptavidin-HRP polymers (1 mg / ml) were pre-diluted 1:10 (4 μl + 36 μl incubation buffer). A final incubation solution was prepared by diluting 1: 300 in an incubation buffer (0.1% BSA in PBS) to a final concentration of 0.33 μg / ml.

100 μl of the resulting solution was added to each well and incubated for 1 hour at room temperature.

The buffer was then removed and the plates were washed 5 times manually with 200 μl wash buffer per well.

Substrate incubation

TMB substrate was balanced to 25 ° C for 1 hour in the dark.

100 μl of substrate solution was added to each well.

ELISA plates were incubated at 25 ° C in the dark for exactly 15 minutes. Then the reaction was stopped by adding 80 μl of 2.5 M H ₂ SO ₄ .

5 minutes after termination of the reaction, OD 450 nm was determined. After evaluating the results, the OD value for each sample was determined.

Evaluation of the results

To establish the adequacy of the samples, the OD values of all samples for gamma-catenin must exceed a certain threshold value, which will confirm the presence of a minimum number of epithelial cells (see example 5).

To detect dysplastic cells, the OD values for HPV16 E7 must exceed a certain threshold value, which will confirm the presence of a minimum number of transformed cells. This threshold value depends on the conditions used in the ELISA, and in the format of the test described by the authors, it was OD = 0.7.

When comparing OD values for HPV 16 E7, gamma-catenin detection in 34 samples with corresponding thresholds revealed that 33 samples contained epithelial cells.

Although the present invention has been described above in preferred embodiments, it will be apparent to those skilled in the art that various modifications may be made thereto without departing from the scope of the invention.

Claims (51)

1. A method for diagnosing a patient’s medically relevant condition, comprising the steps of (a) obtaining an initial sample containing cells taken from a patient or their debris; (b) obtaining a solution of the specified source sample; (c) detecting levels of one or more relevant markers that are indicative of said medically relevant condition in said sample solution; (d) detecting levels of one or more normalization markers that correlate with at least one of the following normalization parameters: the presence or absence of cells of a particular type among the cells present in the sample solution; the presence or absence of a specific nature of the differentiation of cells present in the sample solution; the presence or absence of specific proliferative properties of cells represented in the sample solution; (e) normalizing the detected levels of said one or more relevant markers in at least one of said normalization parameters; and (f) diagnosing a medically relevant condition based on the levels of relevant markers normalized to normalization parameters. 2. The method according to claim 1, where the medically relevant condition is a condition characterized by a property selected from the group consisting of the following properties: the presence or absence of a cell of a particular type in the sample; the presence or absence of a specific nature of cell differentiation in the sample; and the presence or absence of specific proliferative properties of cells in the sample. 3. The method according to claim 2, where the specified medically relevant condition is a disease. 4. The method according to claim 3, where the specified disease is a disorder associated with cell proliferation, a malignant tumor or a lesion preceding them. 5. The method according to claim 4, where the specified malignant tumor is cancer of the head and neck, cancer of the respiratory tract, cancer of the gastrointestinal tract, cancer of the skin and its derivative, cancer of the central and peripheral nervous system, cancer of the urinary system, cancer of the reproductive system, cancer anogenital tract, cancer of the endocrine system, cancer of soft tissues and bones, or cancer of the lymphopoietic and hematopoietic system. 6. The method according to claim 1, where the specified source sample is blood, secretion, smear, aspirate, lavage, sputum, saliva, feces, bile, cells, tissue, biopsy or physiological fluid. 7. The method according to claim 6, where the specified sample contains cells of eukaryotic or prokaryotic organisms. 8. The method according to claim 1, where one or more relevant markers are selected from the group consisting of protein regulators of the cell cycle; metalloproteinases; transmembrane proteins; calcium binding proteins; growth factors; marker molecules that are a sign of viral infections; cell proliferation markers; markers associated with DNA replication; tumor marker proteins; and nucleic acids encoding the corresponding proteins. 9. The method of claim 8, wherein said tumor marker proteins are selected from the group consisting of cyclin-dependent kinase inhibitors, p53, pRb, p14ARF, cyclin E, cyclin A, cyclin B, MN, her2 / neu, mdm-2, bcl -2, EGF receptor, MCM2, MCMZ, MCM4, MCM5, MCM6, MCM7, CDC2, CDC6, protein kinase CDC7, protein phosphatase CDC14, Dbf4, PCNA, Ki67, KiS1, Id1, osteopontin, GRP, renal dipeptidase II and TG. 10. The method according to claim 9, where the cyclin-dependent kinase inhibitor is selected from the group consisting of p16 ^INK4a , p13.5, p14, p15, p19, p21, p27. 11. The method of claim 8, where the indicated marker molecules, which are a sign of viral infections, are a viral protein or a viral nucleic acid. 12. The method according to claim 11, where the viral protein or viral nucleic acid is an HPV protein or nucleic acid derived from an HPV gene selected from the group consisting of HPV L1, HPV L2, HPV E1, HPV E2, HPV E4, HPV E5 , HPV E6 and HPV E7. 13. The method according to claim 1, where one or more normalization markers are selected from the group consisting of cell surface proteins, housekeeping genes; receptor proteins; glycoproteins and / or proteoglycans; carbohydrate structures specific for glycoproteins and / or proteoglycans; cell cycle regulatory proteins; metalloproteinases; transmembrane proteins; calcium binding proteins; growth factors; cell differentiation markers; and proteins associated with DNA replication. 14. The method according to item 13, where one or more markers of normalization are epithelial antigen, cytokeratin or CD antigen. 15. The method according to item 13, where one or more normalization markers are selected from the group consisting of glycoprotein, proteoglycan and a carbohydrate structure present on these molecules. 16. The method according to item 13, where one or more of the normalization markers are enzymes involved in the biosynthesis of glycoproteins and / or proteoglycans. 17. The method according to claim 1, where the detection of relevant markers or markers of normalization is carried out using at least one probe that specifically recognizes these marker molecules and binds to such molecules. 18. The method according to 17, where at least one probe is detectably labeled. 19. The method of claim 18, wherein the probe is labeled with a radioisotope, a bioluminescent compound, a chemiluminescent compound, an electroluminescent compound, a fluorescent compound, a metal chelate, an enzyme, or a biologically relevant binding structure. 20. The method according to claim 19, where the biologically relevant binding structure is biotin, streptavidin or digoxygenin. 21. The method according to 17, where the specified at least one probe is a binding agent. 22. The method according to item 21, where the specified binding agent specifically binds to a marker polypeptide. 23. The method of claim 21, wherein the binding agent is an antibody, antibody fragment, miniantibody, or peptidomimetic containing an antigen binding epitope. 24. The method according to 17, where the specified at least one probe is a lectin containing a carbohydrate-binding site, or a carbohydrate specifically recognized by lectin. 25. The method according to 17, where the specified at least one probe is a nucleic acid molecule that is complementary or anti-complementary to a marker nucleic acid, and where the specified probe specifically hybridizes with the specified marker nucleic acid. 26. The method according A.25, where the specified detection includes a quantitative or semi-quantitative amplification reaction. 27. Test kit for the diagnosis of a medically relevant condition, including (a) at least one reagent for detecting at least one marker molecule, which is a sign of a medically relevant condition; and (b) at least one reagent for detecting at least one normalization marker correlating with at least one of the following parameters: the presence or absence of a particular type of cell or the nature of differentiation in the cells present in the sample solution; the presence or absence of specific proliferative properties of cells represented in the sample solution; and (c) a buffer for solubilization of the samples. 28. The test kit according to item 27, where at least one reagent is fixed on the solid phase. 29. The test kit according to item 27, which further includes at least one of the following components: (a) at least one marker molecule used for positive control reactions and selected from the group consisting of (i) relevant marker molecules that are indicative of medically relevant conditions; (ii) normalization marker molecules correlating with at least one of the following parameters: the presence or absence [of signs] of the specific nature of differentiation in the cells present in the sample solution; the presence or absence of specific proliferative properties of cells represented in the sample solution; and (b) reagents and buffers commonly used to carry out the detection reaction. 30. The test kit of claim 27, wherein the reagents for detecting marker molecules include binding agents specific for said marker molecules and / or nucleic acid probes hybridizing with nucleic acids encoding said marker molecules. 31. The test kit according to claim 30, wherein said binding agents are an antibody, a mini-antibody, or a peptidomimetic containing an antigen-binding epitope. 32. The test kit according to claim 27, wherein said test kit is a diagnostic test kit, a research kit, or an analytical kit. 33. A method for the diagnosis of cervical dysplasia, cervical cancer and previous stages in samples of the human cervix, where the method provides (a) obtaining a solution from a sample of the human cervix; (b) detecting the level of at least one relevant marker, which is a sign of cervical dysplasia; (c) detecting the level of at least one normalization marker indicating the presence of epithelial cells; (d) normalizing the levels of relevant markers by the levels of normalization markers detected in the sample solution; (e) establishing a diagnosis of cervical dysplasia based on the levels of relevant markers normalized to normalization markers. 34. The method according to p, where at least one relevant marker, which is a sign of cervical dysplasia, is selected from the group consisting of p16 ^INK4a , HPV-associated marker, p14ARF, p19, p21, p27, pRb, p53, cyclin E , cyclin A, cyclin B, MN, her2 / neu, mdm-2, bcl-2, EGF receptor, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, CDC2, CDC6, protein kinase CDC7, protein phosphatase CDC14, Dbf4, PCNA , Ki67, KiS1, Id1, osteopontin, claudin-1, GRP, renal dipeptidase and TGFβII receptor. 35. The method according to p, where at least one normalization marker indicating the presence of epithelial cells is selected from the group consisting of gamma-catenin, Ep-Cam, E-cadherin, alpha-catenin, beta-catenin, desmoplakin, hKLK.13, SCCA, uPA1, involucrin, CK8, CK18, CK10, CDK13, vimentin, concanavalin A receptor and lectins. 36. The method according to claim 33, wherein said method is used for early detection or in preliminary screening tests for lesions in the cervix. 37. The method according to p, where the specified sample of the human cervix is a smear, secretion, aspirate, lavage, cells, tissue, biopsy or physiological fluid. 38. The method of claim 33, wherein said epithelial cells are ectocervical cells or endocervical cells. 39. The method of claim 38, wherein the normalization marker indicating the presence of endocervical cells is selected from the group consisting of Ep-Cam, CK8, or CK18. 40. The method according to § 38, where the normalization marker indicating the presence of ectocervical cells is selected from the group consisting of gamma-catenin, E-cadherin, alpha-catenin, beta-catenin, CK13, p120 or involucrin. 41. Test kit for the diagnosis of cervical dysplasia, including (a) at least one reagent for detecting at least one marker molecule that is a sign of cervical dysplasia, selected from the group consisting of p16 ^INK4a , p14ARF, cyclin E, cyclin A, cyclin B, MN, her2 / neu, mdm -2, bcl-2, EGF receptor, mcm-2, mcm-5, claudin-1, markers of infection with human papillomavirus infection, pRb and p53; and (b) at least one reagent for detecting at least one normalization marker indicating the presence or absence of epithelial cells selected from the group consisting of CK8, Ep-Cam, CK13, CK8, CK18, E-cadherin, alpha-catenin , beta-catenin, gamma-catenin or involucrin. 42. The test kit according to paragraph 41, where at least one reagent is fixed on the solid phase. 43. The test kit according to paragraph 41, which further includes at least one of the following components: (a) at least one marker molecule used for positive control reactions and selected from the group consisting of (i) relevant marker molecules that are indicative of cervical dysplasia; (ii) normalization marker molecules correlating with at least one of the following parameters: the presence or absence of cylindrical epithelial cells; the presence or absence of squamous epithelial cells; and (b) reagents and buffers commonly used to carry out the detection reaction. 44. The test kit according to paragraph 41, wherein the reagents for detecting marker molecules include binding agents specific for said marker molecules and / or nucleic acid probes hybridizing with nucleic acids encoding said marker molecules. 45. The test kit according to item 44, where the binding agents are an antibody, a mini-antibody, or a peptidomimetic containing an antigen-binding epitope. 46. The test kit according to paragraph 41, where the test kit is a diagnostic test kit, a device for in vitro diagnostics, a kit for scientific research or an analytical kit. 47. A test kit for the detection of cervical intraepithelial neoplasia containing (a) a reagent for detecting p16 ^INK4a ; and (b) a gamma-catenin detection reagent. 48. The test kit according to item 47, which further includes a reagent for the detection of EP-Cam. 49. The test kit according to item 47, which further includes a buffer for lysis of the sample. 50. The test kit according to clause 47, where the reagents for detection of p16 ^INK4a and gamma-catenin are fixed on solid phases. 51. The test kit according to item 47, which is a device for in vitro diagnostics.

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